Despite enormous recent progress in structural biology in general, determining the structure of membrane proteins has remained difficult. Similarly, we understand very little about the mechanism by which proteins interact with membranes. Here I propose to study this process using site-directed spin labeling (SDSL). SDSL has become a powerful new technique for determining structure and conformational dynamics in soluble and membrane proteins. SDSL is not limited to a particular protein size and can be used to monitor conformational changes in real time under physiological conditions. The primary model system for these studies will be annexin 12, a member of the annexin family of membrane binding proteins. Annexin 12 has three distinctly different states: (1) a water-soluble state of known structure, (2) a Ca2tdependent, peripherally membrane-bound form, and as we recently discovered, (3) a transmembrane form that is lipid and pH dependent, but does not require Ca2+ It is the goal of this proposal to investigate the structures of the peripheral and integral membrane-associated states and determine the factors that modulate the reversible interconversion between them. In addition, we expect this structural work to provide an important foundation to evaluate and rationalize the numerous membrane-related functions of annexins. Sequence analysis suggests that other proteins also could insert into membranes using an annexin-like mechanism. Because of intriguing physiological implications, we will study the membrane interaction of one of these proteins, the estrogen receptor alpha.